Genome-wide in-silico analysis of ethylene biosynthesis gene family in Musa acuminata L. and their response under nutrient stress

Ethylene is a gaseous phytohormone involved in plants’ growth and developmental processes, including seed germination, root initiation, fruit ripening, flower and leaf senescence, abscission, and stress responses. Ethylene biosynthesis (EB) gene analysis in response to nitrogen (N) and potassium (K) stress has not yet been conducted in Musa acuminata (banana) roots. The genome mining of banana (Musa acuminata L.) revealed 14 putative 1-aminocyclopropane-1-carboxylate synthase (ACS), 10 1-aminocyclopropane-1-carboxylate oxidase (ACO), and 3 Ethylene overproducer 1 (ETO1) genes. ACS, ACO, and ETO1 proteins possessed amino acid residues ranging from 422–684, 636–2670, and 893–969, respectively, with molecular weight (Mw) ranging from 4.93–7.55 kD, 10.1–8.3 kD and 10.1–10.78 kD. The number of introns present in ACS, ACO, and ETO1 gene sequences ranges from 0–14, 1–6, and 0–6, respectively. The cis-regulatory element analysis revealed the presence of light-responsive, abscisic acid, seed regulation, auxin-responsive, gibberellin element, endosperm-specific, anoxic inducibility, low-temperature responsiveness, salicylic acid responsiveness, meristem-specific and stress-responsive elements. Comprehensive phylogenetic analyses ACS, ACO, and ETO1 genes of Banana with Arabidopsis thaliana revealed several orthologs and paralogs assisting in understanding the putative functions of these genes. The expression profile of Musa acuminata genes in root under normal and low levels of nitrogen and potassium shows that MaACS14 and MaACO6 expressed highly at normal nitrogen supply. MaACS1 expression was significantly upregulated at low potassium levels, whereas, MaACO6 gene expression was significantly downregulated. The functional divergence and site-specific selective pressures on specific gene sequences of banana have been investigated. The bioinformatics-based genome-wide assessment of the family of banana attempted in the present study could be a significant step for deciphering novel ACS, ACO, and ETO1 genes based on genome-wide expression profiling.


Gene structure analysis and conserved motifs recognition
The genomic and coding sequences of identified genes were retrieved from the database to investigate the intron/ exon arrangement of banana ACS, ACO, and ETO genes.These sequences were further used to draw the gene structure using Gene Structure Display Server (GSDS v2.0 36 ) (available at http:// gsds.cbi.pku.edu.cn/).Multiple EM for Motif Elicitation (MEME) programs (http:// meme.nbcr.net/ meme/) 37 was used to analyze the concluded protein sequences of the banana ACS, ACO, and ETO-1 with a maximum number of motif set as 20.

Gene duplication and calculation of Ka and Ks substitution rates
Putative Gene pair was generated using the knowledge obtained from phylogenetic, motif, and domain analysis of M. acuminata ACS, ACO, and ETO1 genes.These gene pairs were used to calculate the Ka and Ks values through tbtools 38 .The CDS, protein sequence, and gene pair of MaACS, MaACO, and MaETO were utilized.Using Ka/Ks ratio, molecular evolutionary rates of each gene pair were calculated.The Ka/Ks ratio less than 1 indicates the viability of purifying selection; however, Ka/Ks ratio greater than 1 indicates positive selection, and Ka/Ks = 1 indicates neutral selection 39,40 .Using the formula "t = Ks/2λ", with λ (6.05 × 10 −9 ), gene pair divergence was estimated to represent neutral substitution.The MaACS, MaACO, and MaETO like genes were mapped on scaffold using TBtool, and duplicated genes were connected on scaffold using red lines.

Promoter analysis
To analyze the organ-specific expression profile of MaACS, MaACO and MaETO at various development stages, the cis-regulatory element evaluation of recognized banana ACS, ACO, and ETO1 genes was performed by recovering 1000 base pairs sequence upstream from the starting site of banana genomic sequences (promoter region) using the Phytozome database.An ample number of cis-regulatory elements were analyzed in all the Musa acuminata 14 ACS, 10 ACO, and 3 ETO genes by employing the PlantCare database (http:// bioin forma tics.psb.ugent.be/ webto ols/ plant care/ html/) 41 .

Expression analysis
For expression profiling, Reads Per Kilo bases per Million mapped reads (RPKM) values from RNA-seq data were log 2 transformed, and the p-value was calculated using t test.Expression patterns with hierarchical clustering are displayed in Heatmap Illustrator in TBtools 38 .

Putative microRNA target sites analysis
The micro-RNA (miRNA) datasets of Musa acuminata were retrieved from a knowledge-based database called Plant miRNA Encyclopedia (PmiREN, http:// www.pmiren.com/) 42 .There, to find out the miRNAs that target the MaACS, MaACO, and MaETO genes, CDS sequences of all MaACS, MaACO, and MaETO genes were searched for the complementary sequences of miRNAs with the help of psRNA Target (https:// plant grn.noble.org/ psRNA Target/ analy sis? funct ion=3) 43 with default parameters.

Ethical approval
It has been confirmed that the experimental samples of plants, including the collection of plant material, complied with relevant institutional, national, and international guidelines and legislation with appropriate permissions from Authorities of Department of Horticulture, Faculty of Agricultural Sciences, University of the Punjab New campus, Lahore, Pakistan.

Identification of the ACS, ACO and ETO1 genes in banana
ACS, ACO, and ETO genes, involved in the ethylene biosynthesis pathway, were detected in Banana Genome database.Accordingly, the initial analysis predicted 26, 18, and 8 protein sequences of ACS, ACO, and ETO, respectively.The proteins encoded by the same gene isoforms and proteins containing truncated ACS, ACO, and ETO DNA-binding domains were manually removed.Finally, 14 MaACS, 10 MaACO, and 3 MaETO nonredundant genes were identified and used for further analysis.The number of ACS genes was more than that of ACO and ETO.

Comparative phylogenetic relatedness of banana ACS, ACO and ETO1 gene family with Arabidopsis
To investigate the evolutionary relationships between MaACS, MaACO and MaETO1 TFs and Arabidopsis thaliana, a neighbor-joining (NJ) phylogenetic tree was constructed by aligning their full-length protein sequences.The results showed that 14 MaACS proteins were distributed among 3 groups named I, II A, II B, and III (Table S1 and Fig. 1A).
The results for ETO1 depicted that 3 MaETO1 proteins were distributed among 3 groups named I, II, and III (Table S2 and Fig. 1B).
Proteins of common clade usually seem to show similarity in structure and functioning.So, all the ACS, ACO, and ETO1 like proteins of similar Clades may have similar structures and functions.

Gene structures and recognition of conserved motifs and domain
The organization of exon and intron helps verify the evolutionary relationship between genes or organisms 48 .Their distribution patterns and numbers are an evolutionary mark for a gene family.A comprehensive demonstration of Banana genes' exon-intron structures and phylogenetic revealed that the gene structure pattern was consistent with the phylogenetic analysis.The number of introns varied from 3 to 8 in MaACS gene, and no gene in MaACS is intron-less (Fig. 2A, Table S4).
According to Fig. 2B and Table S5, group I contain MaETO1 and MaETO2 genes consisting of 4 exons and 3 introns, while MaETO3 in group III has 4 exons and 3 introns.Similarly, MaACO1 and MaACO2 of group I contain 4 exons and 3 introns, while MaACO10 of group I have 2 exons and 1 intron.MaACO5 of group II comprises 2 introns and 3 exons, while MaACO6 contains 4 exons and 3 introns.MaACO3, MaACO8, and MaACO9 of group III contain 4 exons and 3 introns, while MaACO4 has 3 exons and 2 introns, and MaACO7 consists of 7 exons and 6 introns (Fig. 2C, Table S6).
The conserved motif analysis also verified the classification of MaACS genes.All MaACS protein sequences were loaded into the MEME analysis tool to identify the conserved motifs.As a result, twenty conserved motifs were observed, which were statistically significant with E-values less than 1 × 10 −40 (Fig. 3).The motifs of MaACS proteins identified by MEME were between 15-50 amino acids in length.Motif 1, Motif 2, Motif 5, Motif 6, Motif 7 and Motif 9 are common in all groups.Group I and Group IIA had similar motif patterns.Motif 3 is also common in all groups except in AtACS-12_Type 2 gene of group IIB.All groups (I, IIA, IIB and III) contain M-4 and M-8 other than Group III (MaACS11) and G-I (MaACS3), respectively.Meanwhile, IIB group members have relatively complex motif patterns compared with Group IIA (Fig. 3A).
In MaETO proteins, all the motifs are common in all groups.Motif pattern is also the same in all groups.Figure 3B shows that MaETO gene structures are similar to the corresponding AtETO gene structures.TPR-1 domain is only present in AtETO genes.BTB_POZ domain is only present in MaETO3 genes, similar to atEOL-1 genes.

Gene duplication of banana ACS, ACO and ETO1 genes
The date of duplication of the gene was also estimated through MEGA-X using pairwise alignment that provided Ks and Ka values and then Ka/Ks was calculated manually (Fig. 4).Ks depicts the number of synonymous substitutions per synonymous site, whereas Ka shows the number of nonsynonymous substitutions per nonsynonymous site and the ratio of nonsynonymous (Ka) versus synonymous (Ks) mutation was represented by Ka/Ks.The speculative date for gene duplication of the paralogous group MaACS7_MaACS4 was calculated to be 99.11Mya,acuminata had a Ka/Ks ratio greater than 0.12, suggesting the possibility of considerable functional divergence after the duplication process (Fig. 4A, Table S7).The date of gene duplication for ETO of M. acuminata was calculated 17.26 Mya for paralogous group MaETO2_MaETO1 (Table S8).Similarly, the gene duplication date for MaACO was calculated in 44 paralogous pairs.The segmental duplication date of banana was estimated from 0.45 to 24.13 Mya for paralogous pairs 38 and 14.In all paralogous groups of MaACO the Ka/Ks ratio is greater than 1.15 (Fig. 4B, Table S9).

Analysis of cis-regulatory elements
The spatial-temporal transcriptomic expression of genes is affected by the presence and organization of various cis-regulatory elements at the binding site of transcription factors on the promoter region.In-silico analysis of cis-regulatory elements can be employed to evaluate the putative functions of genes.Cis-regulatory elements related to vital physiological processes such as response to light, seed-specific, endosperm-specific, hormonespecific, meristem-specific, and stress were observed (Fig. 5).Mainly, 9 out of 14 MaACS, all 3 MaETO and 5 out of 10 MaACO genes possess element involved in light responsiveness, 6 MaACS genes possess a fragment of a conserved DNA module that takes part in light responsiveness, and 9 MaACS, 2 MaETO and All 10 MaACO elements involved in the abscisic acid response.1 MaACS and 1 MaETO gene possess elements involved in salicylic acid responsiveness, while 3 MaACS genes showed elements that show response in defense and stress, and 3 MaACS and 10 MaACO genes are related to meristem expression, 3 MaACS and 1 MaETO gene possess factors involved in low-temperature responsiveness, 4 MaACS, and 3 MaETO genes possess Auxin-responsive element, 2 MaACS, and 2 MaETO genes showed element specific to anoxic inducibility, 1 MaACS genes possess elements specific to seed regulation, 4 MaACS genes contain elements involved in endosperm expression, 6 MaACS, and 1 MaETO gene possess elements involved in element involved in gibberellin response, 6 MaACO genes essential for the anaerobic induction and all 10 MaACO genes involved in MeJA-responsiveness.The cis-regulatory

General miRNA expression dataset of Musa acuminata
Heat map for the expression profile of Musa acuminata genes in root under normal and low nitrogen and potassium shows that MaACS14 and MaACO6 expressed highly at a normal nitrogen supply level.Still, they do not respond significantly at low nitrogen levels.MaACO5, MaACO3, MaACO8 show very slight expression at normal nitrogen levels, while MaACS14 express slightly at low nitrogen levels.On the other hand, MaACS1 expresses well at low levels of potassium, whereas MaACO6 gives a significant response at normal levels of potassium.
MaACS4, MaACO4 and MaACO5 show slight behavior in response to low potassium.(Fig. 6).The expression of each banana gene in roots is explained in Table 1.The mature miRNAs sequences were retrieved from Plant MicroRNA Encyclopedia database.Later, those miRNAs that could potentially target MaACS genes were identified with the help of the psRNA Target online tool (https:// plant grn.noble.org/ psRNA Target/ analy sis).Consequently, 49 miRNAs were found, targeting 8 out of 14 MaACS genes.The remaining 6 MaACS genes were not targeted by any of these miRNAs (Table 4).The number of miRNAs targeting these genes varies from 1 to 20 miRNAs per MaACS gene.MaACS 4, 9, 13, 14 are the genes targeted by only 1 miRNAs.On the other hand, MaACS 14 is targeted by 2 miRNAs.3 miRNAs target MaACS 4. None of the gathered miRNAs targeted the remaining 6 MaACS genes.So, this indicates that MaACS 4 is the gene targeted by the maximum number of miRNAs.While discussing ba sed on groups, Group II A was targeted the most by these miRNAs, which was targeted by 3 miRNAs.On the other hand, Group A was targeted by only 3 miRNAs, which is the least among all (Table 4, Table S13).
In MaACO, there are total 32 miRNAs that targeted 6 MaACO genes out of 10.The number of targeting miR-NAs varies from 1 to 17. MaACO5 targeted 2 miRNAs, and the remaining 4 genes were targeted by 1 miRNA.So, this depicts that MaACO5 is the gene targeted by a maximum number of miRNAs (Table 4, Table S14).In ETO of banana, there are two 2 targeted miRNA for both MaETO1 and MaETO2.The total number of targets is 11, the maximum number of miRNA targets on the MaETO1 gene (Table 4, Table S15).

Discussion
The coordination of genes, hormones, and environmental factors made the ripening process successful 57 .Genes like ACS, ACO and ETO involved in ethylene biosynthesis pathway 5,6 perform key role in ripening 58 .Banana genome database (https:// phyto zome-next.jgi.doe.gov/ info/ Macum inata_ v1) implied to identify 14 ACS genes, 10 ACO genes and 3 ETO genes (Table 1) at the genome-wide level.The 14 banana ACS genes were classified into 4 subfamilies (Group I, IIA, IIB, III), whereas in ETO and ACO, there were divided into 3 subgroups (Group I, II, III) using the phylogenetic analysis (Fig. 1).The exon-intron structure and prediction can also be used as evidence for understanding the evolutionary relationships among genes or organisms 48,59,60 .The predicted exon-intron association revealed that all 14 genes have introns (Fig. 2A).The number of introns varied from 3 to 9 in MaACS gene (Fig. 2B), 3 to 4 in MaETO genes (Fig. 2B) and 1-6 in MaACO genes of banana (Fig. 2C).In general, it shows that banana MaACS genes in the same group share similar exon-intron structures (Fig. 2).Exon-intron having similar structures have also been noticed in Arabidopsis, rice and soybean 61,62 which suggest that these structures are evolutionarily preserved.
Motif analysis shows that Motif 3 is common in all groups except in AtACS-12_Type 2 gene of group IIB.While, IIB group members have relatively complex motif patterns compared with Group IIA (Fig. 3A). Figure 3B shows that MaETO gene structures are similar to the corresponding AtETO gene structures.BTB_POZ domain is only present in MaETO gene that was vital for AtETO genes (Fig. 3B).Furthermore, MaACO7 has motifs and PLN02299 domain repeats, similar to CoACO1 and CoACO2 in Camellia oleifera 63 .PLN02403 domain is only present in MaACO5 and MaACO6 (Fig. 3C).The distribution of motifs among ACS proteins (Fig. 3) indicates evolutionary and structural relationships as deduced by the phylogenetic tree [64][65][66] .The motif data analysis by MEME (Fig. 3), and domain analysis using NCBI CDD distinct motifs were identified that were differentially distributed among MaACS (Fig. 3).Meanwhile, at least one or two conservative motif types and spatial distributions in MaACS were present in the same subfamily while some differences were present, implying certain functional similarities of banana ACS members within the same subfamily.In addition, MaACS genes showed structural conservation in subfamilies and were consistent with other plants such as Arabidopsis, rice, cotton and chickpea 61,[67][68][69][70] .In addition, as predicted by in silico analyses, 3 deduced MaACS harbored NLSs to help localize them to the nucleus, but subcellular localization analysis using online tool WoLF PSORT (https:// wolfp sort.hgc.jp/), supposed nucleolus localization in almost all MaACS protein except MaACS 12 and MaACS 14.
The ratio of Ka/Ks provides an understanding of the selection pressure on substituting amino acids.Less than one ratio of Ka/Ks (Ka/Ks < 1) suggests the possibility of purifying selection, whereas more than one ratio of Ka/Ks (Ka/Ks > 1) suggests the likelihood of positive selection 39,71,72 .Generally, evaluation of selective pressure  provides a particular lead for amino acid sequence altered in a protein and is also necessary for interpreting functional residues and protein shifts 73 .Ka/Ks ratios of the sequences from the different banana MaACS groups vary remarkably, while in ETO, only one gene showed Ka/Ks ratio.Despite the differences, all the estimated values of Ka/Ks were less than 1, suggesting that all the ACS sequences in each group undergo strong purifying selection pressure and positive selection might have acted on only a few sites during the process of evolution.In MaACO, all the estimated values of Ka/Ks which were more than one (> 1) shows the possibility of existence of significant positive selection after duplication (Fig. 4).Heat map for the expression profile of Musa acuminata genes in root under normal and low nitrogen and potassium shows that MaACS14 and MaACO6 expressed highly at normal nitrogen supply.Whereas, MaACS1 represents well at low potassium levels, whereas MaACO6 gives a significant response at normal levels of

Conclusion
A comprehensive analysis of genes (ACS, ACO, and ETO1) involved in ethylene biosynthesis in the Musa acuminata (Banana) genome was discussed in this study.The 15 genes of ACS, 10 genes of ACO and 3 genes of ETO1 were categorised into subgroups.The structural and functional properties of each MaACS, MaACO and MaETO member were characterized under Nitrogen (N) and Potassium (K) stress in plant roots, where, MaACO6 expressed highly at both normal and low level of N and P, and MaACS14 expressed well at low nitrogen level, while, MaACS4, MaACO4 and MaACO5 responded to low potassium.Most genes were involved in root cell formation and maintaining the regulation mechanism between roots and stem, suggesting their role in plant root growth and development.The detailed computational inspection of Banana ACS, ACO and ETO proteins revealed in the current study might be selected for cloning purposes at the molecular level, portraying gene expression and studying their interaction with different transcription factors.

Figure 2 .
Figure 2. Phylogenetic relationship and gene structure of ACS, ETO and ACO genes from M. acuminata.

Figure 4 .
Figure 4. Time of gene duplication estimated for different paralogous pairs of MaACS (A) and MaACO (B) genes based on Ks and Ka values.

Figure 5 .
Figure 5. Different cis-acting elements in putative MaACS (A), MaETO (B) and MaACO (C) promoters which are associated with abiotic stresses, hormone responses, growth and development.

Figure 6 .
Figure 6.The heat map shows the expression profile of the M. acuminata ACS (B) and ACO (D) genes in root under the normal level of nitrogen (CR) and low level of nitrogen (NR) and ACS (A) and ACO (C) genes in roots under the normal level of potassium and low level of potassium in different organs.The expression levels of MaACS and MaACO genes are revealed by different colors, which increase from green to red.

Table 1 .
Information about 14 MaACS genes discovered from the genome of M. acuminata, their orthologue in Arabidopsis and their functional annotation.

Table 2 .
Information about 10 MaACO genes discovered from the genome of M. acuminata, their orthologue in Arabidopsis and their functional annotation.

Table 3 .
Information about 10 MaACO genes discovered from the genome of M. acuminata.

Table 4 .
56RNA targets prediction of MaACS(A), MaACO(B) and MaETO(C).Induced by cold but greatly repressed by heat stress49Regulates transition of Arabidopsis root stem cells by transit-amplifying cells to form a regulatory circuit by repressing GRF50The second most prevalent miRNA in NC, neural and ectoderm tissue but was not expressed in blastula55Significantly downregulated under cold stress and it was found to target genes encoding polyphenol oxidase (PPO) and copper-containing proteins56 Vol.:(0123456789) Scientific Reports | (2024) 14:558 | https://doi.org/10.1038/s41598-023-51075-3